The long-term objective of this research proposal is to understand the structural basis for the regulation and activation of Ire1 and its role in stimulating the unfolded protein response (UPR). Ire1 is a transmembrane protein similar in domain structure to mammalian growth-factor receptor kinases and is conserved from yeast to mammals. Ire1 resides in the endoplasmic reticulum (ER) or inner nuclear membrane with its amino-terminal end positioned in the lumen of the ER and its carboxy-terminal end, which contains a protein kinase domain and a ribonuclease domain (RNase) in the cytoplasm. Ire1 acts as a sensor/signaling molecule that is capable of detecting and responding to ER stress. Through genetic studies on the yeast homolog, it has been determined that Ire1 oligomerizes following the accumulation of unfolded proteins in the ER. Oligomerization results in trans-autophosphorylation between Ire1 molecules. This trans- autophosphorylation event is necessary to activate the RNase activity of Ire1 to propagate the UPR signal. The domain architecture of Ire1 represents a novel class of receptor kinases which signals not through downstream phosphorylation events, but through the activation of its adjoining RNase domain. X-ray crystallographic analysis of the cytoplasmic portion containing the kinase and RNase domains will be used to propose a structural basis for the repression and control of RNase activity and the interplay between the kinase and RNase domains. Structural information gained from this study may lead to a new mechanism of enzyme regulation and activation through previously unknown intramolecular interactions with a protein kinase catalytic domain.